Analytical Performance of an Alternative Wavelength in the O-CPC Calcium Assay

The o-cresolphthalein complexone (O-CPC) method is widely used for serum calcium measurement but may exhibit analytical limitations related to background absorbance and bias, particularly at low calcium concentrations. This study evaluated the analytical performance of an alternative wavelength (524 nm) compared with the conventional 572 nm measurement.

Spectral analysis of the calcium-O-CPC complex identified a secondary absorbance feature near 536 nm. Because this wavelength was not available in routine analysers, 524 nm was selected as the nearest operational alternative for analytical evaluation. Performance was assessed using multipoint calibration models, control sera, precision analysis, and comparison with an automated analyzer in 40 samples, including prepared mixtures to extend the analytical range. Agreement between methods was evaluated using regression and Bland-Altman analyses.

Reagent blank absorbance was lower at 524 nm compared with 572 nm (0.048 vs 0.122). Linearity was high at both wavelengths (R²≈0.998). Precision analysis demonstrated comparable CV% values between wavelengths. Regression analysis showed a slope closer to unity and a smaller intercept at 524 nm relative to 572 nm. Bland-Altman analysis demonstrated comparable overall agreement between methods at both wavelengths. In the low concentration subgroup (<9 mg/dL), mean negative bias was reduced from -0.55 mg/dL at 572 nm to -0.30 mg/dL at 524 nm.

Measurement at 524 nm demonstrated comparable overall analytical performance to the conventional 572 nm wavelength and was associated with reduced negative bias in the low concentration range. These findings suggest that alternative wavelength selection may represent a practical approach for optimizing analytical performance in routine O-CPC calcium assays.